Detection of low-abundance proteins and/or detection of proteins from small quantity samples can be a major challenge when performing western blotting. The abundance of the target may be low because the protein itself is expressed at low levels within the source or is difficult to extract (and thus recovery is low), the sample is limited (leaving a scarce overall volume available to load onto the gel), or because of a combination of multiple conditions. The outcome in such situations is often faint or undetectable band(s) during the imaging step, resulting in inconclusive data analysis. This often leads to repeat experimentation and/or re-optimization of conditions, both of which waste time and resources. In this article, we will discuss specific methods from sample preparation through immunodetection that can help overcome challenges and improve signal to noise of low-abundance proteins for more successful western blot detection.
Increasing sensitivity for low abundance targets
When detecting a low abundant target in a western blot, achieving strong signal intensity is critical for making scientific conclusions. Several key factors can help achieve high-quality detection of low-abundant targets:
Efficient protein extraction
Not all proteins are easily extracted, and inefficient extraction can lead to low yields, leading to inability to detect the target in downstream western blotting. Protein extraction techniques should vary depending on the source of the starting material and the location of the protein of interest within the cell. Obtain high protein yield from tissues, cells, or subcellular fractions using reagents that are optimized for mammalian, bacterial, yeast, insect (baculovirus), and plant samples. To prevent protein loss, use broad-spectrum protease inhibitors to protect your proteins during extraction and lysate preparations.
Overview of sample types and recommended protein extraction reagents and kits
|Sample type||Goal||Recommended Thermo Scientific reagents or kits|
|Primary cultured or mammalian cells or tissues||Total protein extraction|
|Cultured mammalian cells or tissues||Subcellular fractionation or organelle isolation|
|Bacterial cells||Total protein extraction|
|Yeast cells||Total protein extraction|
|Insect cells (baculovirus)||Total protein extraction|
|Plant tissue (leaf, stem, root, flower)||Total protein extraction|
Effective protein separation
Figure 1: Find the right gel for your research needs based on the molecular weight of your target protein. The target protein being should migrate through about 70% of the length of the gel for the best resolution.
Optimal separation of low abundant targets is essential to ensuring detection of your target protein by allowing it to be fully accessible to antibody binding during immunoblotting steps. The key to getting optimal separation is choosing the right gel for your target protein. The choice of whether to use one chemistry or another depends on the size of the protein you’re separating. As a general rule, the proteins being targeted should migrate through about 70% of the length of the gel for the best resolution (Figure 1). For separation of a broad range of proteins, two chemistries—Bis-Tris and Tris-glycine—are well suited. However, the alkaline pH of a Tris-glycine gel can cause protein modifications. Bis-Tris gels can provide greater sensitivity for protein detection compared to Tris-glycine gel chemistry. Bis-Tris gel chemistry preserves protein integrity by having a neutral-pH formulation, minimizing protein modification or degradation, resulting in better band resolution.
When studying high or low molecular weight proteins use Tris-Acetate or Tricine gels respectively. High molecular weight proteins will be compressed into a narrow region at the top of Tris-glycine and Bis-Tris gels. By using a Tris-Acetate gel, the target protein can migrate further through the gel. This increased resolution leads to increased transfer efficiencies and higher sensitivity. Low molecular weight proteins when run on Bis-Tris or Tris-Glycine can migrate too close to the bottom of the gel, limiting their resolution (Figure 2). Using a Tricine gel can help ensure proteins migrate within the optimal range of the gel and be fully accessible during immunoblotting steps.
Figure 2: Tricine gels provide better resolution of low molecular weight proteins. A Novex 16% Tricine gel resolved the 17 and 19 kDa bands of cleaved caspase-3, where a Tris-glycine gel did not provide such resolution.
Complete protein transfer
Gel chemistry and transfer method both play critical roles in the quality of gel-to-membrane transfer efficiency. Choosing the right gel is a key factor in the successful transfer of your target protein. A popular general-use gel is a 4-20% Tris-glycine gel, which can effectively separate a mixed range of proteins. However, if studying high molecular weight proteins, the proteins will be compressed into a narrow region at the top of the gel which can decrease the transfer efficiency (Figure 3). Focusing on gels that provide optimal separation of the protein will aid in transfer. In addition, neutral-pH gels such as Bis-Tris and Tris-Acetate can provide better transfer efficiencies than alkaline Tris-glycine gels. The neutral-pH environment minimizes protein degradation and allows for cleaner release of the proteins out of the gel. Preservation of protein integrity is particularly important when separating and transferring low-abundance proteins.
Several transfer methods can be used to successfully transfer and ultimately detect low abundant proteins. Traditional wet transfer (wet tank) offers high efficiency transfers, but at the cost of time, and hands-on effort. Handling inconsistencies are more likely to occur with this more traditional do-it-yourself transfer stack setup- affecting efficiency. Semi-dry blotting provides more convenience and time savings but can have slightly lower efficiency when transferring larger molecular weight proteins (>300 kDa). Dry electroblotting offers both high quality transfers combined with speed and convenience. The ready-to-use stacks used in dry blotting eliminates the need for premade buffers or soaking filter paper and membranes- minimizing handling that can lead to inconsistent performance. The use of a copper anode in the dry blotting system does not generate oxygen gas as a result of water electrolysis, resulting in increased transfer consistency providing performance equal to or better than wet transfer.
Figure 3: Improved transfers of high molecular weight proteins enhances western detection sensitivity. Western blot analysis of EGFR from A431 lysates was transferred from an Invitrogen NuPAGE 3-8% Tris-Acetate Mini Gel, and an Invitrogen Novex 4-20%, Tris-Glycine Mini Gel, using the Invitrogen iBlot 2 Gel Transfer Device. The use of a Tris-Acetate gel improved detection sensitivity of EGFR.
Specificity of antibodies
To achieve strong signals from your target protein, focus on antibodies for which the supplier provides target-specific verification and validation to ensure the antibodies bind to the right target. In addition, use antibodies designated specifically for western blotting or that list western blotting as an application. This will help ensure high-quality performance and specificity to your target protein.
Figure 4: Invitrogen antibody validation for use in western blotting. Antibody Anti-ABCG1 specificity was verified in several cell lines using western blot analysis. Western blot was performed using Anti-ABCG1 Recombinant Rabbit Monoclonal Antibody (Cat. No. MA5-24857). The blot was further probed with a Goat anti-Rabbit IgG (H+L) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A27036). Chemiluminescent detection was performed using SuperSignal West Atto Ultimate Sensitivity Substrate (Cat. No. A38556) and imaged on the iBright Imaging System.
Figure 5: Invitrogen antibody specificity for target antigen. Anti-ABCG1 was verified by cell treatment to ensure that the antibody bound to the antigen stated. Altered expression of proteins upon cell treatment demonstrates antibody specificity. Western blot using ABCG1 Recombinant Rabbit Monoclonal Antibody (2A10) (Cat. No. MA5-24857), shows increased expression in THP-1 cells upon treatment with TPA (24h) seen in Lane 2, TPA (24h) along with IL-33 (24h) seen in Lane 3 and TPA (24h) along with TNF-alpha (48h) as seen in Lane 4 as compared to un-treated THP-1 cells as seen in Lane 1. Cell treatment validation info.
Increase low-end detection sensitivity
To achieve the highest sensitivity, a chemiluminescent HRP (horseradish peroxidase) conjugated system should be used. Chemiluminescence yields the greatest potential sensitivity of any available detection method for western blotting. HRP is relatively small, which enables more molecules conjugated per IgG molecule, providing greater sensitivity over AP (alkaline phosphatase) systems. Furthermore, advancement and improvements in chemiluminescent substrates for HRP have enabled even higher sensitivity over other detection systems for western blotting. Chemiluminescent systems when optimized can provide the ability to detect down to the attogram level.
As with other components in the western blotting systems, there are many chemiluminescent substrate choices available. High sensitivity substrates such as SuperSignal West Atto Ultimate Sensitivity Substrate, provide the most sensitive detection, delivering over 3x more sensitivity than conventional ECL substrates (Figure 6). SuperSignal West Atto is an ultrasensitive enhanced chemiluminescent (ECL) substrate that enables protein detection down to the high-attogram. It is the ideal choice for detection of very low-abundance targets or when using precious samples that require maximum levels of sensitivity (Figure 7).
Figure 6: Cell lysates of various cell lines were loaded at 30 µg/lane in NuPAGE 3-8% Tris Acetate gel and electrophoresed at 150V for ~90 minutes at 4⁰ C. Gels were first equilibrated with 20% ethanol then transferred to nitrocellulose membranes using the iBlot2 Gel Transfer Device (IB21001) with iBlot2 Transfer Stacks (IB23001). Membranes were blocked in 5% milk in 1X PBST diluted with UltraPure DI water for 1 hour at room temperature. Mucin-1 mouse monoclonal (MA1-90954) was diluted in 5% milk in 1X PBST at 1:1K dilution then incubated overnight at 4°C with continuous gentle rocking. Goat anti mouse HRP secondary antibody was diluted in 2.5% milk in 1X PBST at 1:10K. Membranes were image on the iBright Imaging System. Blots were exposed for 5 second with SuperSignal West Atto and 3 minutes with Pierce ECL Chemiluminescent Substrate.
Increased detection of low abundance proteins with high sensitivity substrates. Mis-folding and hydrophobicity of membrane-bound or nuclear expressed proteins, such as Mucin 1 (MUC1), can be difficult to extract, resulting in lower abundance during western blot detection. Mucin 1 is a cell surface protein that acts as an adhesion and anti-adhesion protein by forming mucous membranes in epithelial cells. This protein is highly O-glycosylated and moderately N-glycosylated to yield functional mature mucin. Glycosylation degree varies in mucin accounting for 50-90% of its total weight. Therefore, depending on tandem repeats and degree of glycosylation MUC1 can weigh between 250 and 500 kDa. Both SuperSignal West Atto and Pierce ECL were used to detect mucin in various cell lines with various degrees of MUC1 glycosylation. SuperSignal West Atto Ultimate Sensitivity Substrate was able to detect the less abundant glycosylation of MUC1 above 460 kDa and below 200 kDa compared to Pierce ECL Substrate.
Detection of target proteins in low protein loads. Low target abundance can be the result of low total sample yield. With a lower than anticipated protein yield and limited sample available overall, a higher sensitivity system may be needed to adequately detect the specific target.
Learn more about chemiluminescent western blot detection
Figure 7: SuperSignal West Atto Ultimate Sensitivity Substrate and SuperSignal West Femto was used to detect Ampka alpha and Erk1/2 in HeLa cell lysates. HeLa cell lysates starting at 0.20 µg/µL was loaded in Novex 4-20% Tris-Glycine gel (WXP42020BOX) and separated at 225V for ~40 minutes. Gels were transferred to nitrocellulose membranes using the Power Blotter System (PB0012) and Power Blotter Select Stacks (PB3310). Membranes were blocked in 5% milk in 1X TBST for 1 hour at room temperature. AmpK alpha mouse monoclonal (MA5-14922) and Erk1/2 Mouse monoclonal (2696; Cell Signaling Technology) antibodies were diluted in 5% milk in TBST at 1:1K then incubated overnight at 4°C with continuous gentle rocking. Goat anti-Mouse conjugated HRP (31430) was diluted in 5% milk in TBST at 1:50K dilution of a 1 mg/mL stock. Membranes were image on the iBright Imaging System.
When it comes to detecting the signal, digital imaging systems will provide the greatest dynamic range and ease of determining optimal exposure to detect low abundant proteins. With advances in camera technology and developments in imaging software, the limitations of traditional signal capture with X-ray film have become increasingly evident. CCD (charged coupled device) cameras are based on light-sensitive silicon chips that convert photons to digital signals. Improvements in chip design have enabled the development of sensitive, cooled-CCD-based cameras with higher light-capturing performance than X-ray film. Powerful high-resolution cooled-CCD cameras in instruments such as the iBright Imaging Systems, enable the capture and analysis of western blots with greater sensitivity, linearity and dynamic range than X-ray film. X-ray film displays only 1.5 orders of dynamic range compared to iBright Imaging Systems and other equivalent 16-bit CCD cameras, which display at least a 4-fold dynamic range (Figure 8). This larger dynamic range permits the capture of strong chemiluminescent signals without sacrificing detection of faint bands. Reference Luminometer plates show evidence of a greater dynamic range of signal using the iBright Imaging System over traditional X-ray film (Figure 8).
Figure 8. Image sensitivity and dynamic range comparison of film and iBright Imaging System. (A) At a binning of 3x3, images of a Reference Luminometer plate on the iBright Imaging system shows greater sensitivity than those from film. (B) Image analysis of the Reference Luminometer plate images demonstrates a greater dynamic range of signals using the iBright Imaging System compared to film.
For Research Use Only. Not for use in diagnostic procedures.